Mutation in collagen II alpha 1 isoforms delineates Stickler and Wagner syndrome phenotypes.

PURPOSE
Stickler syndrome is an arthro-ophthalmopathy with phenotypic overlap with Wagner syndrome. The common Stickler syndrome type I is inherited as an autosomal dominant trait, with causal mutations in collagen type II alpha 1 (COL2A1). Wagner syndrome is associated with mutations in versican (VCAN), which encodes for a chondroitin sulfate proteoglycan. A three-generation Caucasian family variably diagnosed with either syndrome was screened for sequence variants in the COL2A1 and VCAN genes.


METHODS
Genomic DNA samples derived from saliva were collected from all family members (six affected and four unaffected individuals). Complete sequencing of COL2A1 and VCAN was performed on two affected individuals. Direct sequencing of remaining family members was conducted if the discovered variants followed segregation.


RESULTS
A base-pair substitution (c.258C>A) in exon 2 of COL2A1 cosegregated with familial disease status. This known mutation occurs in a highly conserved site that causes a premature stop codon (p.C86X). The mutation was not seen in 1,142 ethnically matched control DNA samples.


CONCLUSIONS
Premature stop codons in COL2A1 exon 2 lead to a Stickler syndrome type I ocular-only phenotype with few or no systemic manifestations. Mutation screening of COL2A1 exon 2 in families with autosomal dominant vitreoretinopathy is important for accurate clinical diagnosis.

highly variable, even within a family [5]. Autosomal recessive forms of Stickler syndrome may present with ocular features, such as high myopia and vitreoretinal degeneration but no presenile cataract, and systemic features may include but are not limited to short stature, hearing impairment, facial structural abnormalities but no midline clefting, and causal gene-dependent spondyloepiphyseal dysplasia [7].
The expression of the COL2A1 gene, associated with STL1, is tissue-dependent, due to an alternative splicing of exon 2, which results in two isoforms of type II collagen [18]. Type IIB is the shorter form with 53 exons (exon 2 spliced), and is mainly expressed in the cartilage while type IIA is the longer form with 54 exons (including exon 2) and is predominantly present in the vitreous [18]. Thus, mutations in COL2A1 exon 2, such as a premature stop codon, may lead to the ocular variant of the STL1. This ocular condition shares typical STL1 ocular features, such as membranous vitreous and radial perivascular retinal degeneration, but shows none or few systemic manifestations [3,[19][20][21]. To date, several families harboring COL2A1 exon 2 mutations have been reported [3,19,20,[22][23][24][25]. In these families, the reported penetrance of vitreoretinal degeneration is more than 90% in affected patients at the age of 20 [19,20].
Wagner syndrome (OMIM 14200) is also associated with the extracellular matrix component gene versican (VCAN; alternatively called CSPG2), identified in 2005 by Miyamoto et al., which encodes for a large chondroitin sulfate proteoglycan versican [26][27][28][29][30]. We confirmed the involvement of this gene in 2009 by describing an intronic base pair splice site substitution in VCAN segregating with the disease in a multigenerational family resulting in a truncated protein affected by the splicing [31]. Wagner syndrome clinical features involve ocular manifestations close to those found in Stickler syndrome. However, patients also complain of nyctalopia, where the vitreous phenotype is different as characterized by an optically empty aspect with avascular strands and veils or fibrillary condensations [32], retinal detachments occur rarely, and retinal features demonstrate retinitis pigmentosa-like "bone spicule" pigmentary atrophy [20] with accompanying electroretinogram abnormalities. Nevertheless, in some cases, distinguishing Wagner syndrome from the ocular-only variant of STL1 may be difficult. Erosive vitreoretinopathy syndrome (OMIM 143200) is another ocular-only disorder, first reported by Brown et al. [33], that can also lead to indeterminate diagnosis based on clinical features only [30]. The clinical features of erosive vitreoretinopathy syndrome include night blindness, visual field defects, and chorioretinal atrophy. The variability in the clinical presentations of vitreoretinal disease phenotypes underscores the importance of careful clinical assessment.
We clinically and genetically report a three-generation Caucasian family from the southeast United States demonstrating AD vitreous degeneration with variable phenotypes among affected members. Over the years, one affected member was initially diagnosed clinically with Stickler syndrome and then Wagner syndrome by her ophthalmologist. We conducted Sanger sequencing of the COL2A1 (NM_001844) and VCAN (NM_004385) genes to delineate the genetic etiology of disease in this family.

METHODS
Study subjects: Ten individuals (six affecteds, four unaffecteds) from a three-generation Caucasian family was recruited. All consenting family members (four males, six females) were recruited under the approval of the Duke University Institutional Review Board according to the principles of the Declaration of Helenski, under the research protocol entitled "Clinical and Molecular Analysis of Genetic Eye Disorders", to include molecular genetic testing (protocol number Pro00008040). Individuals underwent ophthalmic examinations that included health histories regarding systemic issues such as cleft palate, midline defects, skeletal or joint abnormalities, and early onset arthritis. The clinical evaluation included assessment tests of Early Treatment Diabetic Retinopathy Study visual acuity (Snellen equivalent) and intraocular pressure, slit-lamp inspection of the anterior segment, and indirect ophthalmoscopy to inspect the fundus [34,35].
Genomic DNA was extracted using AutoPure LS® DNA Extractor and PUREGENE™ reagents (Gentra Systems Inc., Minneapolis, MN) from blood or saliva samples. DNA samples were also collected from 1,142 unrelated ethnically matched Caucasian healthy control participants.
Gene screening and sequence analysis of collagen type II alpha 1 and versican genes: Primers for PCR and sequencing were designed to cover coding and untranslated gene regions, including intron-exon boundaries, using the ExonPrimer and Primer3 programs (Helmholtz Zentrum, Munich, Germany). Primers were selected to produce amplified product sizes not to exceed 900 bp for optimal sequence output and reading. Large exons or untranslated gene regions were covered with overlapping amplicons, with a minimal 50 bp of overlapped sequence. All 54 COL2A1 exons and 15 VCAN exons were examined. Appendix 1 displays the optimized primer sequences used for VCAN and COL2A1 screening.
Genomic DNA of two affected individuals (II:2 and II:3; Figure 1) of the study family was initially screened for sequence variations in the COL2A1 and VCAN genes. The DNA of the remaining family members was subsequently screened to determine and confirm sequence variants segregation.
PCR was conducted using an Eppendorf Mastercycler Pro S® with a standard touchdown PCR protocol. PCR amplicons were visualized with 2% agarose gel electrophoresis. BigDye™ Terminator 3.1 was used to perform sequencing reactions, and ABI3730XL robotics was used to process the DNA fragments (Applied Biosystems Inc. [ABI], Foster City, CA). The Sequencher® 5.0 Software (Gene Codes, Ann Arbor, MI) was used to analyze the base pair calls. Sequences of affected and unaffected individuals were aligned to a known reference genomic sequence (UCSC Genome Browser) and compared for sequence variation. Sorting Intolerant From Tolerant (SIFT) [36] and Polymorphism Phenotyping (PolyPhen2) [37] software tools were used to predict mutational consequence of all COL2A1 and VCAN variations segregating with disease.
Genotyping: Applied Biosystems (ABI) TaqMan® SNP Genotyping assays were designed and employed to measure the allelic frequencies in 1,142 ethnically matched control DNA samples. Variants of interest were screened with PCR assay technology using TaqMan probes according to the manufacturer's protocol (Applied Biosystems). Alleles were detected and allelic discrimination were analyzed with ABI Prism® 7900HT Sequence Detection System and ABI Sequence Detection Systems 2.4 software, respectively (Applied Biosystems). For quality control, positive and negative controls were run in the same experiment.

Complementary deoxyribonucleic acid tissue expression:
We investigated the expression of the COL2A1 messenger ribonucleic acid (mRNA) construct(s) in fetal ocular tissues to verify the presence of type IIA and/or type IIB isoforms. Fetal ocular tissue panels not affected with disease were established internally by acquisition of whole eye globes from Advanced Bioscience Resources (Alameda, CA). Twentyfour-week fetal eyes were obtained and preserved in RNAl-ater® Foster City, CA within minutes of abortion and shipped overnight on ice. Whole globes were dissected the same day as they arrived, and specific ocular tissues were isolated by snap-freezing the samples and storing at −80 °C until RNA extraction. RNA was extracted from each tissue sample independently using the Ambion Foster City, CA mirVana Total RNA Extraction Kit per the protocol. The tissue samples were homogenized in Ambion's lysis buffer using an Omni Bead Ruptor 24 Homogenizer per protocol. Reverse transcription reactions were performed with Invitrogen's SuperScript™ III First-Strand Synthesis kit to obtain cDNA (Life Technologies, Grand Island, NY).
In-house fetal eye cDNA was amplified using primers that spanned multiple exons, not to exceed 600 bp (Appendix 1). PCRs were run using a standard protocol. Visualization of the PCR products was done on a 2% agarose gel through electrophoresis at 120 V for 50 min. Products with exon 2 were expected to amplify at 510 bp, whereas those that did not contain the exon 2 were expected to produce a product of 303 bp. Band extraction, purification, and sequencing of the bands were conducted to verify the amplicon.

RESULTS
Clinical features: A three-generation family with affected member variable ocular-only diagnosis of either Stickler or Wagner syndrome was ascertained. The disease appeared to be transmitted in an AD inheritance pattern and showed variable expressivity with 100% penetrance (Figure 1). Six affected and four unaffected individuals participated in the study. Clinical data were obtained where available.
The proband, patient III:3, was a 40-year-old woman with moderate myopia and history of retinal detachment in the left eye (OS) at the age of 18 years. Periodically, she underwent prophylactic peripheral laser photocoagulation treatment bilaterally. She had cataract extraction surgery of both eyes at the age of 37 (right eye, OD) and 40 (OS). Fundus examination showed bilateral vitreous syneresis, with a vitreous Tissue expression: We examined COL2A1 expression across normal fetal eye tissues to verify the presence or absence of type IIA and/or type IIB isoforms (Figure 3). Both COL2A1 mRNA isoforms (type IIA and type IIB) were expressed in the fetal retina/retinal pigment epithelium and choroid (Appendix 1). The COL2A1 mRNA type IIB isoform (excluding exon 2) was expressed in the sclera, optic nerve, and cornea. Gel extraction and Sanger sequencing of the specific product bands confirmed our findings of the isoforms except for the optic nerve, where the IIA isoform was not sequenced.

DISCUSSION
We report a nonsense mutation in a large Caucasian family consisting of six affected individuals variably diagnosed with Stickler and Wagner syndromes. A base pair change at c.258C>A leading to a premature stop codon in exon 2 of COL2A1 was cosegregated with the disease status. Sequencing of ocular tissues confirmed the presence or  whereas this exon is spliced in the COL2A1 type IIB isoform (B), which is expressed by adult differentiated chondrocytes. Primers were designed to amplify both cDNA isoforms: The COL2A1 cDNA primers span 303 bp when amplifying COL2A1 type IIB cDNA (excluding exon 2), and 510 bp when amplified COL2A1 type IIA cDNA (including exon 2). Ex 1, Ex 2, Ex 3, and Ex 8 depict exons 1, 2, 3, and 8. absence of exon 2, demonstrating that isoforms may be ocular tissue specific. The mutation was not present in more than 2,000 chromosomes, validating the rarity of this mutation and confirming Stickler syndrome has a predominant ocular-only phenotype.
Two striking features of Stickler syndrome are, as in our reported family, the high penetrance and variable expressivity. In the literature, the same mutation as in our family (c.258C>A; NM_001844.4) demonstrated high penetrance [3,19,20]. In families harboring alternative COL2A1 exon 2 mutations, the ocular manifestation penetrance was also high-from 90% [20] to 100% [24,38]. The variable expressivity, even within the same family [39], contrasts with the high disease penetrance: In most reported COL2A1 exon 2 mutations, ocular features were variable as either myopia, retinal detachment [22], or retinal degeneration [23] could be absent in affected patients. Furthermore, two common ocular features in COL2A1 exon 2 mutations are vitreous degeneration and radial perivascular retinal degeneration [3,19,20]. Systemic manifestations were rarely associated with COL2A1 exon 2 mutations, as manifestations were present in few cases [3,19,20,22]. In our family, only one affected individual presented with cleft palate.
Underlying causes of variable expressivity in ocular-only STL1 are still undetermined. However, in recent years the phenotypic variability of exon 2 mutations has been hypothesized to be due to degradation of mRNA by nonsensemediated decay (NMD) or synthesis of alternatively spliced protein [21].
NMD is a regulation pathway involving the targeted degradation of mRNA that contains a premature stop codon. In this way, NMD may play a role in phenotype variability by minimizing the potential damage caused by premature termination codons [40,41]. In achondrogenesis and hypochondrogenesis caused by COL2A1 mutations, a relationship has been proved between the severity of the phenotype and the amount of type II collagen within the cartilage extracellular matrix [42]. This implies that not only qualitative but also quantitative factors likely modulate phenotypes linked to the COL2A1 gene [43], as in haploinsufficiency due to NMD.
Haploinsufficiency due to NMD was reported by Kaarniranta et al. [44], who found that heterozygous inactivation of COL2A1 gene in the murine model led to structural defects and alterations that resulted from haploinsufficiency in ocular tissues containing type II collagen. These alterations included vitreous changes similar to those seen in patients with Stickler syndrome, which included reduced immunostaining of type II collagen in the vitreous and retina, in addition to reduced density of vitreous filaments in COL2A1+/− mutant mice [45]. Furthermore, in COL2A1 exon 2 mutant mice with mutant allele encoding COL2A1 mRNA without exon 2 [46], IIA+/− mutant embryos demonstrated, at an early stage, craniofacial abnormalities of truncated frontonasal structures and hypoplasia of the midface tissues. These malformations were more frequent in IIA−/− mutants. These findings are consistent with COL2A1 type IIA mRNA expression in regions of active recruitment of cells for chondrogenesis and in areas of skeletal growth [41].
Alternatively, a second hypothesis is that nonsensemediated altered splicing can be caused by disruption within the splicing cis element [21]. Minigene constructs created by McAlinden et al. demonstrated that disruptions in the enhancer sites in COL2A1 exon 2 favor the production of the procollagen type IIB isoform. The decrease in the ratio of type IIA compared to type IIB leads to variance in expression levels, perhaps one isoform predominating over another, but the less expressed is not completely absent. These studies highlight the alternative imbalance between the two isoforms, which may have adverse effects during ocular embryogenesis [21].
Systemic manifestations associated with STL1, particularly facial development abnormalities and midline clefting as reported here (individual II:3; Figure 1), have been observed in some cases of COL2A1 exon 2 mutations, with a frequency depending on the series, 1%, 4%, and 43% in the Donoso [3], Parma [19], and Richards [22] series, respectively. These findings are not inconsistent with exclusive expression of the longer type IIA isoform in the adult vitreous [47], as embryonic expression of this isoform has been demonstrated in chondroprogenitor tissues [48].
The Cys86Stop mutation has previously been reported in four families with Stickler syndrome whose genealogy was traced to the 16 th century. Subsequently, Donoso et al. identified a member of the branch that migrated to the southeastern and mid-southern United States during the 19 th century [3,19,20]. Some members of these families included direct descendants from the passengers of the 1620 Mayflower voyage [20] who arrived on the northeastern coast and then migrated to the south. Interestingly, an affected individual (III:2) reviewed her genealogy and traced her ancestry to the state of Georgia. If descendants from Donoso's family indeed migrated south due to the Cherokee Land Grant of 1813, the similar geographical region of their cohort and ours would not exclude the possibility that the families could be related [20]. The apparent founder effect coupled with literature estimations of 50,000 to 100,000 descendants that could be related to the original family with this reported mutation demonstrates the importance of the genotype-phenotype relationship in patients with Stickler syndrome with this particular mutation [3].
The overarching similarities in phenotypes among vitreoretinal diseases make accurate diagnosis difficult clinically. Clinicians must understand and be updated on all allied conditions associated with Stickler syndrome to properly diagnose patients [47]. The characteristics of vitreous and retinal degeneration may guide molecular testing, but in case of doubt, a retinal specialist should be referred [47]. Although concentrations of exon 2 mutations are for predominantly ocular-only phenotypes, family members with mutations can still have systemic manifestations, seen in individual II:3, who presented with a cleft palate at a young age. The broad phenotypic variation seen in families with Stickler syndrome underscores the importance of using clinical and genetic testing to properly diagnose and treat patients with Stickler and Wagner syndromes.

APPENDIX 1. SUPPLEMENTAL SECTION CONTAINS 4 TABLES AND 1 FIGURE.
Supplemental section contains 4 tables and 1 figure. Table  S1 contains all variants identified in COL2A1. Table S2 and S3 contains primers used for COL2A1 and VCAN gene screening. Table S4 contains primers used for cDNA amplification. Figure S1 contains PCR products of COL2A1 cDNA tissue expression. To access the data, click or select the words "Appendix 1."

ACKNOWLEGEMENTS
The authors would like to thank all family members for participation in the study. This research effort was supported by the National Institutes of Health Grant EY014685, The Lew Wasserman Award from Research To Prevent Blindness Inc., and the Duke-National University of Singapore core grant. (TLY) This research was also supported by the Toulouse Hospital Young Researcher Fellowship, the Fondation pour la Recherche Médicale and Fondation de France (VS).